There have been a number of research efforts investigating the role of carbohydrates in physiologically relevant recognition. (See Brandley, B. K., and Schnaar, R. L., J. Leuk. Biol. (1986) 40:97; and Sharon, N., and Lis, H., Science (1989) 246:227). Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. Many oligosaccharide structures can be created through the differential activities of a smaller number of glycosyltransferases. Their diverse structures, then, can be generated with relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell-cell interactions. Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) on interacting cells have been described (see Dodd, J., and Jessel, T. M., J. Neurosci. (1985) 5:3278; Regan, L. J., et al., Proc. Natl. Acad. Sci. USA (1986) 83:2248; Constantine-Paton, M., et al., Nature (1986) 324:459; and Tiemeyer, M., et al., J. Biol. Chem. (1989) 263:1671). Further, the question has been raised as to the nature of the leukocyte receptor for ELAM-1 (see Bevilacqua et al. Proc. Natl. Acad. Sci. USA (1987) 84:9238).
Tumor associated glycolipids have been reported in fetal tissue and a variety of human cancers, including CML cells (Fukuda, M. N., et al., J. Biol. Chem. (1986) 261:2376; Magnani, J. L., et al., J. Biol. Chem. (1982) 257:14365; Hakomori, S., et al., Biochem. Biophys. Res. Comm. (1983) 113:791). This has led to the hypothesis that these structures may be important in many developmental and oncogenic processes (J. L. Magnani et al., J. Biol. Chem. (1982) 257:14365). Smaller quantities of most of these carbohydrates can be found in normal human tissue (see Fukushi, Y., et al., J. Exp. Med. (1984) 160:506), but until now no function for these structures has been reported.
Adhesion of circulating leukocytes to stimulated vascular endothelium is a primary event of the inflammatory response. Several receptors have been implicated in this interaction, including a family of putative lectins that includes gp90.sup.MEL (Leu8), GMP-140 (PADGEM) and ELAM-1 (Gong, J.-G., et al., Nature (1990) 343:757; Johnston, G. I., et al., Cell (1989) 56:1033; Geoffroy, J. S., and Rosen, S. D., J. Cell Biol. (1989) 109:2463; Lasky, L. A., et al., Cell (1989) 56:1045). All three of the presently known selectins have been shown to recognize carbohydrates (see Lasky, Science, 258:964-969, 1992). Endogenous ligands for these receptors are being identified.
ELAM-1 is interesting because of its transient expression on endothelial cells in response to IL-1 or TNF (Bevilacqua, M. P., et al., Science (1989) 243:1160). The time course of this induced expression (2-8 h) suggests a role for this receptor in initial neutrophil extravasation in response to infection and injury. Furthermore, Bevilacqua et al. (see Bevilacqua, M. P., et al., Proc. Natl. Acad. Sci. USA (1987) 84:9238) have demonstrated that human neutrophils or HL-60 cells will adhere to COS cells transfected with a plasmid containing a cDNA encoding the ELAM-1 receptor.
Recently, several different groups have published papers regarding ELAM-1 ligands which are also referred to as LECAM-2 ligands. Lowe et al. (1990) demonstrated a positive correlation between the LECAM-2 dependent adhesion of HL-60 cell variants and transfected cell lines, with their expression of the sialyl Lewis X (sLe.sup.x) oligosaccharide, Neu NAc .alpha.2-3Gal-.beta.1-4(Fuc .alpha.1-3)-GlcNAc and generally shown more specifically as Sia.alpha.2-3Gal.beta.1-4[Fuc.alpha.1-3]-GlcNAc. By transfecting cells with plasmids containing an .alpha.(1,3/1,4) fucosyltransferase, they were able to convert non-myeloid COS or CHO lines into sLe.sup.x -positive cells that bind in an LECAM-2 dependent manner. Attempts to block LECAM-2 dependent adhesion using anti-sLe.sup.x antibodies were uninterpretable due to the agglutination of the test cells by the antibody. They conclude that one or more members of a family of oligosaccharides consisting of sialylated, fucosylated, lactosaminoglycans are the ligands for the lectin domain of LECAM-2. Phillips et al. (1990) used antibodies with reported specificity for sLe.sup.x to inhibit the LECAM-2 dependent adhesion of HL-60 or LEC11 CHO cells to activated endothelial cells. Liposomes containing difucosylated glycolipids with terminal sLe.sup.x structures inhibited adhesion, while those containing nonsialylated Lex structures were partially inhibitory. Walz et al. (1990) were able to inhibit the binding of a LECAM-2-lgG chimera to HL-60 cells with a monoclonal antibody directed against sLe.sup.x or by glycoproteins with the sLe.sup.x structure, but could not demonstrate inhibition with CD65 or CD15 antibodies. Both groups concluded that the sLe.sup.x structure is the ligand for LECAM-2.
Information regarding the DNA sequences encoding endothelial cell-leukocyte adhesion molecules are disclosed in PCT published application WO90/13300 published Nov. 15, 1990. The PCT publication cites numerous articles which may be related to endothelial cell-leukocyte adhesion molecules. The PCT publication claims methods of identifying ELAM-ligands, as well as methods of inhibiting adhesion between leukocytes and endothelial cells using such ligands and specifically refers to MILAs which are described as molecules involved in leukocyte adhesion to endothelial cells.
LECAM-1 is interesting because of its involvement in lymphocytic and neutrophil influx (Watson et al., Nature, 349:164-167 (1991)). It was expressed in chronic lymphocytic leukemia cells which bind to HEV (see Spertini et al., Nature, 349:691-694 (1991)). It is believed that HEV-like structures at sites of chronic inflammation are associated with the symptoms of disease such as rheumatoid arthritis, psoriasis, and multiple sclerosis.
A broad range of ELAM-1 ligands are disclosed in PCT/US91/05416 published as WO 92/02527 (published 20 Feb. 1992) to Brandley et al. and in PCT/US90/02357 published as WO 90/13300 (published 15 Nov. 1990) to Hession et al. both of which are incorporated herein by reference in their entirety and specifically to disclose oligosaccharide structures which reportedly act as ELAM-1 and LECAM-1 ligands.
The selectins are a family of three cell-cell adhesion proteins that mediate various leukocyte-endothelial adhesion events (reviewed in Lasky, L. A., Science, 258:964-969 (1992); McEver, R. P., Curr. Opin. Cell Biol. 4:840-849 (1992); Bevilacqua, M. P., and Nelson, R. M., J. Clin. Invest., 91:379-387 (1993); Rosen, S. D., Semin. in Immunol., 5:237-249 (1993). L-selectin is expressed on the surface of leukocytes and participates in the homing of blood borne-lymphocytes to peripheral lymph nodes (Gallatin, W. M., Weissman, I. L., and Butcher, E. C., Nature, 903:30-34 (1983); Geoffroy, J. S., and Rosen, S. D., J. Cell Biol., 109:2463-2469 (1989)) by mediating attachment to the specialized endothelial lining cells of high endothelial venules (HEV). L-selectin, a lectin-like receptor bearing a calcium-type domain, mediates the attachment of lymphocytes to high endothelial venules (HEV) of lymph nodes (Gallatin et al., Nature, 303:30-34 (1983); Lasky, L. A., Science, 258:964-969 (1992); and Bevilacqua et al., J. Clin. Invest., 91:370-387 (1993)). L-selectin is also involved in the rolling interaction of neutrophils with venular endothelium at certain sites of acute inflammation (Lewinsohn et al., J. Immunol., 138:4313-4321 (1987); Ley, K., Gaehtgens, P., Fennie, C., Singer, M. S., Lasky, L. A., and Rosen, S. D., Blood, 77:2553-2555 (1991); Von Adrian, U., Chambers, J. D., McEvoy, L. M., Bargatze, R. F., Arfors, K. E., and Butcher, E. C., Proc. Natl . Acad. Sci. USA, 88:7538-7542 (1991)), an essential step for the ultimate extravasation of the leukocyte. The other two selectins, E- and P-, are expressed on endothelial cells where they mediate attachment to neutrophils, monocytes and specific subsets of lymphocytes. L-selectin participates in the entry of lymphocytes and monocytes into sites of chronic inflammation (Dawson et al., Eur. J. Immunol., 22:1647-1650 (1992), and Spertini et al., J. Exp. Med., 175:1789-1792 (1992)). The selectins perform their adhesive functions by virtue of C-type lectin domains at their amino termini (Drickamer, K., J. Biol. Chem., 263:9557-9560 (1988)). Reflecting a high degree of sequence similarity among these domains (60-70%), the biological ligands for L-selectin on HEV and for E- and P-selectin on leukocytes share a requirement for sialic acid (reviewed in Varki, A. Curr. Opin. Cell Biol., 4:257-266 (1992)). Moreover, each selectin is capable of recognizing sialyl Lewis X [sLe.sup.x, i.e., Neu5AC.alpha.2.fwdarw.3Ga1.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)/GlcNAc] and related structures (reviewed in Stoolman, L. M., Cell Surface Carbohydrates and Cell Development, pp. 71-97 (1992) (M. Fukuda, ed.) CRC Press, Boca Raton, Fla.), although inhibition studies indicate that these compounds have a low binding affinity. There is evidence, based on the use of carbohydrate-specific antibodies, that sLe.sup.x -related structures are present in the actual biological ligands of the selectins (Phillips, M. L., Nudelman, E., Gaeta, F., Perez, M., Singhal, A. K., Hakomori, S., and Paulson, J. C., Science, 250:1130-1132 (1990); Walz,. G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B., Science, 250:1132-1135 (1990); Polley, M. J., Phillips, M. L., Wayner, E., Nudelman, E., Singhal, A. K., Hakomori, S., and Paulson, J. C., Proc. Natl. Acad. Sci. USA, 88:6224-6228 (1991); Berg, E. L., Yoshino, T., Rott, L. S., Robinson, M. K., Warnock, R. A., Kishimoto, T. K., Picker, L. J., and Butcher, E. C., J. Exp. Med., 174:1461-1466 (1991); Sawada, M., Takada, A., Ohwaki, I., Takahashi, N., Tateno, H., Sakamoto, J., and Kannagi, R., Biochem. Biophys. Res. Commun., 193:337-347 (1993); Nogard, K. E., Moore, K. L., Diaz, S., Stults, N. L., Ushiyama, S., McEver, R. P., Cummings, R. D., and Varki, A., J. Biol. Chem., 268:12764-12774 (1993)). Nonetheless, each selectin has a set of preferred biological ligands (Larsen, G. R., Sako, D., Ahern, T. J., Shaffer, M., Erban, J., Sajer, S. A., Gibson, R. M., Wagner, D. D., Furie, B. C., and Furie, B., J. Biol. Chem., 267:11104-11110 (1992) Berg, E. L., Magnani, J., Warnock, R. A., Robinson, M. K., and Butcher, E. C., Biochem Biophys. Res. Commun., 184:1048-1055 (1992)), although information is lacking on what distinguishes the ligands of one selectin from those of another.
Presently, the best characterized ligands are the HEV-associated ligands for L-selectin, known as GlyCAM-1 (previously termed Sgp50) and Sgp90 (Imai, Y., Singer, M. S., Fennie, C., Lasky, L. A., and Rosen, S. D., J. Cell Biol., 113:1213-1221 (1991)). These endothelial-associated ligands are mucin-like glycoproteins with sulfated, sialylated and fucosylated O-linked oligosaccharide chains and were originally detected by precipitation of lymph node extracts, metabolically labeled with .sup.35 SO.sub.4, with a soluble L-selectin/immunoglobulin chimera. Other lower affinity ligands may exist that fail to be precipitated by the chimera but nonetheless participate in functionally significant interactions in the context of cell-cell binding events (Berg, E. L., Robinson, M. K., Warnock, R. A., and Butcher, E. C. J. Cell. Biol., 114:343-349 (1991)). GlyCAM-1 is released into conditioned medium of cultured lymph nodes as an intact molecule (Lasky, L. A., Singer, M. S. Dowbenko, D., Imai, Y., Henzel, W. J., Grimley, C., Fennie, C., Gillett, N., Watson, S. R., and Rosen, S. D., Cell, 69:927-938 (1992); Brustein, M., Kraal, G., Medius, R. E., and Watson, S. R., J. Exp. Med., 176:1415-1419 (1992)), suggesting that it is a secreted product and/or a loosely associated peripheral membrane component. In contrast, Sgp90 is an integral membrane protein, requiring detergent for extraction (S. Hemmerich and S. Rosen, unpublished results). Molecular analysis has revealed GlyCAM-1 to be a novel mucin-like glycoprotein, and more recently Sgp90 has also been shown to be an HIV-specific glycoform of the mucin CD34, Baumhueter, S., Singer, M. S., Henzel, W., Hemmerich, S., Renz, M., Rosen, S. D. and Lasky, L. A., Science, 262:436-438 (1993). GlyCAM-1 and Sgp90 are sulfated, fucosylated, and sialylated glycoproteins (Imai, Y., and Rosen, S. D., Glycoconjugate J., 10:34-39 (1993)). The O-linked chains of GlyCAM-1 have been shown to be heterogeneous in both size and charge. Some of the chains bear multiple charges, the major contribution apparently coming from sulfation rather than sialylation. The interaction of both GlyCAM-1 and Sgp90 with L-selectin depends on their sialylation, confirming earlier findings that sialidase treatment of lymph node HEV impairs lymphocyte attachment and lymphocyte trafficking (Rosen, S. D., Singer, M. S., Yednock, T. A., and Stoolman, L. M., Science, 228:1005-1007 (1985); Rosen, S. D., Chi, S. I., True, D. D., Singer, M. S., and Yednock, T. A., J. Immunol., 142:1895-1902 (1989)). However, exhaustive desialylation does not completely abrogate-the ligand activity of GlyCAM-1, suggesting that a sialic acid-independent mode of recognition also exists (Imai, Y., Lasky, L. A., and Rosen, S. D. Glycobiology, 4:373-381). The sialic acid which forms part of the ligand binding site of GlyCAM-1 appears to be in an .alpha.2.fwdarw.3 linkage, since the linkage-specific sialidase from Newcastle disease virus partially inactivates GlyCAM-1 as a ligand. Furthermore, both in competitive inhibition studies and direct binding studies, sLe.sup.x -type oligosaccharides manifest ligand activity for L-selectin whereas the Lewis X-type structures with .alpha.2.fwdarw.6 linked Neu5Ac are inactive (Foxall, C., Watson, S. R., Dowbenko, D., Fennie, C., Lasky, L. A., Kiso, M., Hasegawa, A., Asa, D., and Brandley, B. K., J. Cell Biol., 117:895-902 (1992)). An essential contribution from fucose is suspected, since sialyllactose (i.e., Neu5Ac.alpha.2.fwdarw.3Ga1.beta.1.fwdarw.4Glc) as compared to sLe.sup.x is relatively inactive as a competitor of L-selectin binding. Moreover, fucose has been shown to be a critical determinant for the neutrophil ligands for P- and E-selectin (Larsen, G. R., Sako, D., Ahern, T. J., Shaffer, M., Erban, J., Sajer, S. A., Gibson, R. M., Wagner, D. D., Furie, B. C., and Furie, B., J. Biol. Chem., 267:11104-11110 (1992)), and in light of the sequence similarity among the lectin domains of the selectins is likely to be important for L-selectin ligands as well.
The earlier studies have largely focused on which oligosaccharide compounds can act as ligands. In our earlier work, it was determined that attachment of a sulfate moiety to the oligosaccharide compound has a significant effect on the ability of the compound to act as a ligand and thereby developed the invention upon which copending application Ser. No. 07/943,817, filed Sep. 11, 1992, and incorporated herein by reference, was based (see also Imai, Y. and Rosen, S. D., Glycoconjugate J., 10:34-39 (1993); Smai, Y., Lasky, L. A. and Rosen, S. D., Nature, 361:555-557. Evidence has been presented that sialyl Lewis X-related oligosaccharides, i.e., EQU Sia.alpha.2-3Gal.beta.1-4[Fuca1-3]GlcNAc
have ligand activity, albeit very weak, for L-selectin (Foxall et al., J. Cell Biol., 117:895-902 (1992), Berg et al., Biochem. Biophys. Res. Comm., 184.:1048-1055 (1992), and Imai et al., Glycobiology). Based on studies with a carbohydrate-directed antibody, evidence also exists that endogenous HEV-ligands actually possess sialyl Lewis X-related structures (Sawada, M., Biochem. Biophys. Res. Comm., 193:337-347 (1993)). Various sulfated carbohydrates including sulfatide, fucoidin (Imai et al., J. Cell Biol., 111:1225-1232 (1990), related glycolipids (Suzuki et al., Biochem. Biophys. Res. Comm., 190:426-434 (1993) and a sulfated form of Lewis X/a (Green et al., Biochem. Biophys. Res. Comm., 188:244-251 (1992), i.e. EQU SO4-3Gal.beta.1-4/3[Fuca1-3/4]GlcNAc
have all been shown to have ligand activity for L-selectin.
The present application describes that work and provides further elaboration on the details of the carbohydrate structures which are ligands for selectins in general and L-selectin in particular, including the nature of the novel carbohydrate determinants of GlyCAM-1 involved in its recognition of L-selectin.